Hepatitis C virus (HCV) is a major causative agent of parenterally transmitted non-A, non-B hepatitis which is responsible for most cases of acute and chronic liver deseases including cirrhosis and hepatocellular carcinoma. However, progress of HCV research is severely hampered by a lack of an easily available animal model or tissue culture system. Here we propose to develop a tissue culture system to produce high-titer HCV viruses in order to investigate the entry mechanism of HCV infection and pathogenesis. This is an attractive system because it may allow for production of an unlimited, high-titer of HCV pseudotype viruses, and it will provide an efficient and safe approach to HCV entry studies. The three specific aims to be pursued in this proposal are: (1) Development of a tissue culture system to produce high-titer HCV pseudotype viruses. We will utilize murine leukemia virus-based vectors to produce HCV pseudotype viruses. (2) Examination of the host tropism of HCV infection. The HCV pseudotype viruses will be used to infect cell lines from different species and tissues. These results may provide some clues on HCV host tropism and pathogenicity. (3) Identification and characterization of the cellular receptor(s) for HCV. Proteins that have been implicated in HCV entry such as CD81 and human LDL receptor will be tested to investigate whether they can mediate HCV infection using the HCV pseudotype viruses. Furthermore, the HCV pseudotype viruses will be used to identify and characterize HCV receptor or co-receptor. These studies should pave the way to elucidate the entry mechanism of HCV infection and pathogenesis.